• Proceedings of the Korean Society of Applied Pharmacology
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• 1998.11a
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• pp.127-127
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• 1998

Specific inhibitors of a calcium activated neutral protease calpain could be used for the treatment of neurodegenerative diseases, cataract and muscular dystrophy diseases because of their therapeutic effects. In the course of screening for potential calpain inhibitors from microorganisms, a new analogue of chymostatins named calmostinol was isolated from the culture filtrate of an actinomycete. The MW was determined to be 596 [(M + H)$\^$+/] by FAB-MS in glycerol matrix. The structure was elucidated to be N-[((S)-1-carboxy-2-phenylethyl)-carbamoyl]-${\alpha}$-[2- iminohexahydro-4(S)-pyrimidyl]-L-glycyl- L-valyl-phenylalaninol, by the spectroscopic methods such as NMR and MS fragmentation studies. Calmostinol exhibited strong activity against calpain while not against a Ca$\^$2+/ -independent cysteine protease papain.

• Korean Journal of Food Science and Technology
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• v.8 no.4
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• pp.253-259
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• 1976

Two kinds of sepharose-bound pronases were successfully prepared. The immobilized pronase, directly coupled to cyanogen-bromide activated sepharose, retains 22.6% of original specific activity against casein. However, ${\omega}-aminoalkyl$ sepharose immobilized pronases, in which extension arms of ${\omega}-aminoalkyl$ group $(number\;of\;-CH_2-\;is\;8,\;10,\;and\;12)$ are used, retain almost 100% of original specific activity. Studies of enzyme stability, pH dependence, temperature dependence, and Km values are presented.

• Applied Biological Chemistry
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• v.33 no.4
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• pp.337-342
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• 1990

The pretense from Halobacterium sp. was purified by ethanol precipitation and gel filtration on Sephadex G-75 and G-100. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis It's specific activity was 364units/mg protein and yield was 14% of the total activity of the culture filtrate. The Km value against casein was determined to be $4.2{\times}10^{-4}M$ by Lineweaver-Burk plot The optimal temperature and pH for the enzyme activity were $35^{\circ}C$ and pH 8.0, respectively. The enzyme was stable from 5.0 to 11.0 at relatively wide range of pH but was inactivated at the temperature above $50^{\circ}C$. $Ca^{2+}$ and $Mg^{2+}$ appeared to react as activators whereas $Fe^{3+},\;Zn^{2+},\;Cu^{2+},\;Hg^{2+}\;and\;Cd^{2+}$ as inhibitors. The enzyme activity reduced with increasing the concentration of NaCl : the apparent activity with 2M NaCl was 65% as compared with that without the salt However the enzyme was unstable without salts : the activity was lost when dialyzed against distilled water for 2hr, whereas maintained against 0.1M solution of $CaCl_2$ for 6hr.

• Korean Journal of Food Science and Technology
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• v.30 no.6
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• pp.1333-1338
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• 1998

Several enzymes of kimchi ingredients were assayed to improve the product quality using these quality related enzyme information. Among various hydrolases, amylase and protease were selected with respect to lactic acid fermentation. Peroxidase and ascorbic acid oxidase were studied for off flavor production and ascorbic acid destruction. The amount of protein in kimchi ingredients, specific and total enzyme activity of sample were compared. Regarding total enzyme activity of sample, ${\alpha}-amylase$ activity of salted and fermented anchovy, dried red pepper and salted and fermented shrimp were higher than other ingredients. Activity of salted and fermented anchovy was 2,790.0 units/g sample. Salted and fermented anchovy, oyster and Chinese radish showed the highest ${\beta}-amylase$ activity (4.4, 2.1, 1.0 units/g sample, respectively). Salted and fermented anchovy showed the highest protease activity of 13.4 PU/g sample, followed by salted and fermented shrimp and dried red pepper. For peroxidase, Chinese radish, cucumber, green onion showed the highest activity of 7.2, 6.8 and 5.6 units/g sample, respectively. In case of ascorbic acid oxidase, salted and fermented anchovy showed the strongest enzyme activity (331.4 units/g sample), followed by dried red pepper and salted and fermented shrimp.

• The Korean Journal of Food And Nutrition
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• v.13 no.4
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• pp.348-352
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• 2000

The stabilization of wheat $\beta$-amylase( Himaltosin GL, Hankyu-Bio) was attained by modification wish periodate-oxidized soluble starch. The specific activities of modified enzyme at pH 9.7 and pH 8.0 were 17% and 96%, respectively, compared with that of native enzyme. The pH stability of modified enzyme was increased at pH 2~5 and 6~12 in the presence of $\alpha$-cyclodextrin( $\alpha$-CD) compared with that of native enzyme, and optimum pH of the enzyme was changed from pH 5.0 to pH 7.0 by the modification. Thermal stability of the modified enzyme was increased. After treatment at 6$0^{\circ}C$ for 10min, the activity remained 8% for the enzyme modified at pH 8.0 in the presence of $\alpha$-CD and tested in the presence of $\alpha$-CD, 5% for the native enzyme. The native enzyme and modified enzyme showed one peak in HPLC. The molecular weight of the modified enzyme was slightly increased in HPLC analysis.

• Biotechnology and Bioprocess Engineering:BBE
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• v.1 no.1
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• pp.46-50
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• 1996

In order to design industrial scale reactors and proceises for multi-phase biocatalytic reactions, it is essential to understand the mechanisms by which such systems operate. To il-lustrate how such mechanisms can be modeled, the hydrolysis of the primary ester groups of triglycerides to produce fatty acids and monoglycerides by lipased (glycerol-ester hydrolase) catalysis has been selected as an example of multiphase biocatalysis. Lipase is specific in its behavior such that it can act only on the hydrolyzed (or emulsified) part of the substrate. This follows because the active center of the enzyme is catalytically active only when the substrate contacts it in its hydrolyzed form. In other words, lipase acts only when it can shuttleback and forth between the emulsion phase and the water phase, presumably within an interphase or boundary layer between these two phases. In industrial applications lipase is employed as a fat splitting enzyme to remove fat stains from fabrics, in making cheese, to flavor milk products, and to degrade fats in waste products. Effective use of lipase in these processes requires a fundamental understanding of its kinetic behavior and interactions with substrates under various environmental conditions. Therefore, this study focuses on modeling and simulating the enzymatic activity of the lipase as a step towards the basic understanding of multi-phase biocatalysis processes.

• BMB Reports
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• v.34 no.1
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• pp.21-27
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• 2001

The structural stability of brain pyrydoxine-5'-phosphate (PNP) oxidase and the catalytic properties of the monomeric species were investigated. The unfolding of brain pyridoxine-5'-phosphate (PNP) oxidase by guanidine hydrochloride (GuHCl) was monitored by means of fluorescence and circular dichroism spectroscopy Reversible dissociation of the dimeric enzyme into subunits was attained by the addition of 2 M GuHCl. The perturbation of the secondary structure under the denaturation condition resulted in the release of the cofactor FMN. Separation of the processes of refolding and reassociation of the monomeric species was achieved by the immobilization method. Dimeric PNP oxidase was immobilized by the covalent attachment to Affi-gel 15 without any significant lass of its catalytic activity. Matrix-bound monomeric species were obtained from the reversible refolding processes. The matrix bound-monomer was found to be catalytically active, possessing only a slightly decreased specific activity when compared to the refolded dimeric enzyme. In addition, limited chymotrypsin digestion of the oxidase yields two fragments of 12 and 161 kDa with a concomitant increase of catalytic activity The catalytically active fragment was isolated by ion exchange chromatography and analyzed for association of two subunits using the FPLC gel filtration analysis. The retention time indicated that the catalytic fragment of 16 kDa behaves as a compact monomer. Taken together, these results are consistent with the hypothesis that the native quaternary structure of PNP oxidase is not a prerequisite for catalytic function, but it could play a role in the regulation.

• Microbiology and Biotechnology Letters
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• v.27 no.4
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• pp.298-303
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• 1999

An $\alpha$-D-galactosidase ($\alpha$-D-galactoside galactohydrolase, EC 3. 2. 1. 22) from earthworm was purified by affinity chromatography using N-$\varepsilon$-aminocaproyl-$\alpha$-D-galactopyranosylamine coupled to sepharose and its properties were examined. The specific activity of the purified enzyme, tested with p-nitrophenyl-$\alpha$-D-galactopyranoside as substrate, was 314 units/mg protein, representing an 122-fold purification of the original crude extract. The final preparation obtained from by Sephadex G-25 chromatography showed a single band on SDS-polyacrylamide gel electrophoresis. The molecular weight was determined to be 48,000 by SDS-polyacrylamide gel electrophoresis. The purified galactosidase was showed maximum activity at pH 4.5 and 4$0^{\circ}C$, and was stable in the pH and temperature ranges from 4.0 to 5.5 and 30 to 5$0^{\circ}C$, respectively. The enzyme activity was inhibited by Zn2+, Hg2+ and Co2+. When the purified $\alpha$-galactosidase treated to guar gum for 6 hour, gel-promoting property was increased. It was clear that enzymatic elimination of galactose from guar gum by purified $\alpha$-galactosidase would lead to a significant increase in gelation ability.

• Journal of Plant Biology
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• v.32 no.4
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• pp.265-274
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• 1989

The effect of Ca2+ on auxin-induced ehtylene production in etiolated mungbean (Vigna radiata W.) hypocotyls was studied. Auxin-induced ethylene production by mungbean seedlings which had been germinated in the presence of 5-10mM Ca2+ (High Ca2+ ; HC) is greater than that by seedlings which had been germinated in distilled water (Low Ca2+ ; LC). The effect of Ca2+ on auxin-induced ethylene production was greatly increased after 12hr of incubation period. The stimulation of auxin-induced ethylene production by Ca2+ was specific, since divalent cations, such as Mg2+ and Mn2+ did not enhance auxin-induced ethylene production. Calcium also promoted ethylene evoluation induced by methionine and 1-Aminocyclopropane-1-carboxylic acid(ACC). The effect of Ca2+ on auxin-induced ethylene production was not caused by increase in free IAA or ACC contents of hypocotyl tissue. Dimethyl sulfoxide and Triton X-100, that disrupts the emembranes, inhibited ethylene production to a greater extent in LC segments than in HC segments. Addition of Ca2+ to the incubation medium for LC segments resulted in enchancement of ethylene production probalby because the membrane integrity is supported under these conditions. Comparison of activity of Ethylene Forming Enzyme(EFE) in LC and HC hypocotyl segments indicated that the enzyme activity of HC was about 2 times higher than that of L.C. It is suggested that Ca2+ increases the activity of plasma membrane-bound EFE through its stabilizing effect onn the membrane, which in turn brings about promotion of ethylene production.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.6
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• pp.375-379
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• 2002

Methyl mercaptan oxidase was successfully induced in Thiobacillus thioparus TK-m using methyl mercaptan gas, and was purified for the detection of mercaptans. The purification procedure Involved a DEAE (diethylaminoethyl) -Sephacel, or Superose 12, column chromatography with recovery yields of 47.5 and 48.5%, and specific activities of 374 and 1240.8 units/mg-protein, respectively, The molecular weight of the purified methyl mercaptan oxidase was 66.1kDa, as determined by SDS-PAGE. The extract, from gel filtration chromatography oxidizes methyl mercaptan, producing formaldehyde, which can be easily detected by the purpald-coloring method. The optimized temperature for activity was found to be at 55$\^{C}$. This enzyme was inhibited by both NH$_4$Cl and (NH$_4$)$_2$SO$_4$, but was unaffected by either KCl or NaCl at less than 200 mM. With K$_2$SO$_4$, the activity decreased at 20 mM, but recovered at 150 mM. In the presence of methanol, full activity was maintained, but decreased in the presence of glycerin, ethanol and acetone 43, 78 and 75%, respectively.